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Abstract:

Disclosed is a method for preparing a furan fatty acid, more particularly
a method for preparing a furan fatty acid by heat-treating
7,10-dihydroxy-8(E)-octadecenoic acid (DOD) in hexane. The present
disclosure provides a simple method for producing 7,10-EODA from a
dihydroxyl fatty acid precursor. Considering the difficulties in
purifying natural furan fatty acids because of easy attack by peroxyl
radicals and small quantity and the complicated multiple steps for
chemical synthesis, the present disclosure provides a useful way to
produce the biologically activity F-acid cost-effectively in large scale.

Claims:

2. The method for preparing a furan fatty acid according to claim 1,
wherein the 7,10-dihydroxy-8(E)-octadecenoic acid is produced by the
microorganism Pseudomonas aeruginosa using oleic acid or vegetable oil
containing oleic acid as substrate.

8. An antioxidant comprising the 7,10-epoxy-octadeca-7,9-dienoic acid
according to claim 7.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119 to
Korean Patent Application No. 10-2011-0049750, filed on May 25, 2011 in
the Korean Intellectual Property Office, the disclosure of which is
incorporated herein by reference in its entirety.

TECHNICAL FIELD

[0002] The present disclosure relates to a method for preparing a furan
fatty acid, and in particular, to a method for preparing a furan fatty
acid including heat-treating 7,10-dihydroxy-8(E)-octadecenoic acid (DOD)
in hexane.

BACKGROUND ART

[0003] Furan fatty acids (F-acids) are a large group of fatty acids
characterized by a furan ring, which carries at one α-position an
unbranched fatty acid chain with 9, 11, or 13 carbon atoms and at the
other α-position a short straight-chain alkyl group with 3 or 5
carbon atoms (1). Mostly two β-positions of the furan ring are
substituted by either one or two methyl residues or other group. F-acid
without any substitutions on both β-positions of the furan ring was
also found in the seed oil of Exocarpus cupressiformis (2). F-acids are
widely distributed in nature as trace components of plants, fishes,
amphibians, reptiles, microorganisms and mammals including human (1,
3-7).

[0004] Although the biological role of F-acids in the biological system is
not fully understood, it has been pointed out that F-acids can be
involved in various important biological functions acting as antioxidant,
antitumoral and antithrombotic (8-10). In some fishes F-acids comprised
up to 25% of the acids in the liver lipids and accumulated during the
spawning season indicating possible correlation between F-acids and the
fertilization process (11). The correlation between consumption of fish
rich in F-acid and protection against coronary heart disease mortality
has been confirmed in several studies (12). F-acid has also been reported
to have inhibitory effects on blood platelets aggregation (9) and to have
potential antitumor activity (8). F-acids were found to prevent oxidation
of linoleic acid (13) and act as antioxidants in plants (14). Some
studies demonstrated that F-acids underwent oxidation by ring opening to
form dioxoenes (15-16) in the presence of linoleic acid as co-substrate
demonstrating that F-acid acted as a radical scavenger (17-18).

[0005] Biosynthesis of F-acids are complicated and quite different from
sources. The biogenetic precursor of the most F-acids is known to be
linoleic acid. It was recognized that plants synthesized the basic
skeleton of F-acids from different sources (19). However, study with the
radio-labeled feeding to fish indicated that fish synthesized neither the
alkyl side chain nor the furan ring of F-acids (1). Therefore F-acids in
fish were considered to be originated from diet, especially algae.
Consequently F-acids are introduced into human body through the diet like
vegetables and fishes. Diet-derived F-acid are incorporated into the
tissue and blood of mammals, especially into phospholipids (20) where
they might act as radical scavengers resulting into inhibition of blood
platelet aggregation (9).

[0006] These reports indicated that F-acid could be an essential
nutritional factor for mammals and could be used as an active component
of functional food. However, no matter what biological sources of F-acids
were, biosynthesis of F-acids required multistep reactions due to the
formation of furan ring and the different alkyl substituents.
Accordingly, chemical synthesis of F-acids required complicated multistep
reactions and chemical catalysts causing difficulties and high costs for
industrial application.

[0007] Recently we have produced 7,10-dihydroxy-8(E) octadecenoic acid
(DOD) from vegetable oil containing oleic acid by microbial conversion
(21). DOD is a dihydroxy monoenoic C18 fatty acid uniquely carrying
two hydroxyl groups at carbon 7 and 10 and a trans double bond between
carbon 8 and 9. Based on unique structural feature, it is highly
plausible to modify DOD molecules by intra- or intermolecular interaction
via chemical or physical ways. In our constant efforts to modify DOD for
biological and industrial applications, we developed a simple way to
produce a novel biologically active F-acid from DOD through one-step heat
treatment.

SUMMARY

[0008] The present disclosure is directed to providing a method for
preparing a novel, biologically active furan fatty acid through a
one-step heat treatment process using 7,10-dihydroxy-8(E)-octadecenoic
acid prepared from vegetable oil by microbial conversion.

[0009] The present disclosure is also directed to providing a novel furan
fatty acid prepared by the method.

[0010] The present disclosure is also directed to providing an antioxidant
containing the furan fatty acid.

[0011] In one general aspect, the present disclosure provides a method for
preparing a furan fatty acid, including: mixing
7,10-dihydroxy-8(E)-octadecenoic acid (DOD) with hexane; and
heat-treating the resulting mixture.

[0012] The 7,10-dihydroxy-8(E)-octadecenoic acid is represented by
Chemical Formula 1:

##STR00001##

[0013] The 7,10-dihydroxy-8(E)-octadecenoic acid may be synthesized
chemically or produced by microorganisms. Specifically, it may be
produced by the microorganism Pseudomonas aeruginosa using oleic acid or
vegetable oil containing oleic acid as substrate.

[0014] The microorganism may be any bacterium belonging to Pseudomonas
aeruginosa. Specifically, it may be Pseudomonas aeruginosa PR3 (NRRL
strain B-18602).

[0015] The Pseudomonas aeruginosa PR3 is deposited in the Agricultural
Research Service Culture Collection (Peoria, Ill., USA) with under
Accession No. NRRL B-18602.

[0016] 7,10-Dihydroxy-8(E)-octadecenoic acid is a hydroxy fatty acid
having two hydroxyl groups on a C18 fatty acid chain, each at carbon
7 and 10, and having a trans double bond between carbon 8 and 9.

[0018] The natural vegetable oil may be prepared by solvent extraction or
pressed extraction that have been traditionally employed to extract oil
from natural plant seeds or fruits or may be easily purchased from the
market.

[0019] Furan fatty acids (F-acids) are a large group of fatty acids
characterized by a furan ring. They are widely distributed in the nature
as trace components in plants, fish, amphibians, reptiles,
microorganisms, and mammals including human. As described in the
Background section, furan fatty acids are important essential nutritional
factors for mammals and can be used as active components in functional
foods.

[0020] The furan fatty acid according to the present disclosure is
7,10-epoxy-octadeca-7,9-dienoic acid (7,10-EODA). Most of the previously
known furanoid fatty acids are found in trace amounts in fish. Mostly,
the furan ring is substituted by one or two methyl residues. The length
of the side chain is various. It is also known to be contained in human
blood in trace quantity. One with no methyl residue is not known to occur
naturally and it is reported that a small amount can be produced as
intermediate when linoleic acid is treated with lipoxygenase. However,
the resulting furanoid fatty acid is different in the position of the
furan ring from that provided by the present disclosure. That is to say,
it has an epoxy structure carbon 10 and 13. As such, the novel furanoid
fatty acid presented in the present disclosure is a new substance that
has never been reported.

[0021] When mixing DOD with hexane, the mixing ratio of DOD and hexane may
be determined appropriately. For efficient mixing with DOD and production
of the desired product, hexane may be used in an amount corresponding to
0.001-1000 times that of DOD. Specifically, 10-1000 μL of hexane may
be used per 10 mg of DOD.

[0022] When preparing the furan fatty acid according to the present
disclosure, the heat treatment may be performed at 30-150° C. for
1-150 hours. Specifically, it may be performed at 90° C. for 36-96
hours. According to experiments performed by the inventors, 7,10-EODA is
produced with very high yield of 70-80% at temperatures between
85° C. and 95° C., and increasing the reaction time gives
the same result as that of elevating the reaction temperature. The
production of 7,10-EODA started at 12-96 hours and reached maximum at
36-96 hours (see FIGS. 6 and 7).

[0023] In another general aspect, the present disclosure provides
7,10-epoxy-octadeca-7,9-dienoic acid prepared by heat-treating
7,10-dihydroxy-8(E)-octadecenoic acid (DOD) in hexane.

[0024] The furan fatty acid prepared in accordance with the present
disclosure is 7,10-epoxy-octadeca-7,9-dienoic acid (7,10-EODA). Whereas
most of the naturally occurring furanoid fatty acids have one or two
methyl groups on the furan ring, the furan fatty acid of the present
disclosure does not have a methyl group. Also, the method for preparing a
furan fatty acid according to the present disclosure allows preparation
of the novel furan fatty acid from DOD through a one-step heat treatment
process, without using a chemical catalyst.

[0025] The inventors of the present disclosure have found out for the
first time that the novel, physiologically active compound can be
prepared from DOD through heat treatment. Chemical analysis of the
purified product revealed that the produced new furan fatty acid was
7,10-epoxy-octadec-7,9-dienoic acid (7,10-EODA) (see FIG. 5 and
Experimental Result 2).

[0026] In another general aspect, the present disclosure provides an
antioxidant including the 7,10-epoxy-octadeca-7,9-dienoic acid
(7,10-EODA). Since the Furan fatty acids are reported to have an
antioxidant activity, as described in the Background section, the
antioxidant activity of 7,10-EODA of the present disclosure was analyzed
by DPPH assay of measuring radical scavenging activity. As a result,
7,10-EODA showed a radical scavenging activity increasing in a
dose-dependent manner. Although the activity was lower than that of
α-tocopherol or ascorbic acid, 7,10-EODA showed a clear radical
scavenging activity in a dose-dependent manner (see FIG. 8). Accordingly,
the furan fatty acid according to the present disclosure can be applied
in various industrial fields as antioxidant having an antioxidant
activity.

DESCRIPTION OF DRAWINGS

[0027] FIG. 1. Analysis of the crude extract obtained from conversion of
DOD by heat treatment. Analysis was carried out by GC (A) and TLC (B).
Major unknown compound in GC analysis is indicated by the arrow in TLC
analysis. Lane 1; standard DOD, lane 2; crude extract of the heat-treated
DOD. Other experimental conditions were given in materials and methods
section.

[0028]FIG. 2. Analysis of the crude extract and the purified unknown
compound obtained from conversion of DOD by heat treatment. Analysis was
carried out by GC (A) and TLC (B). Upper and lower GC chromatogram
represented crude extract and purified samples, respectively. Major
unknown compound in GC analysis is indicated by the arrow in TLC
analysis. Lane 1; standard DOD, lane 2; crude extract of the heat-treated
DOD, lane 3; purified unknown compound. Other experimental conditions
were given in materials and methods section.

[0029]FIG. 3. Electron-impact mass spetrum of the methylated unknown
compound in FIG. 2. Major fragments were indicated by the arrow.
Analytical conditions are explained in materials and methods section.

[0038] 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was produced according
to our previous report (21). In brief, olive oil (1%, v/v) was added as a
substrate to the 24 hr-old culture of Pseudomonas aeruginosa PR3 which
was cultivated aerobically at 28° C., 200 rpm in shaking incubator
followed by an additional 72 hr incubation. Crude DOD extract obtained by
extraction of the culture with an equal volume of ethyl acetate was
applied to the silica-gel column (1.5 cm I.D.×30 cm) for
purification. Fractionation was conducted with two column volumes of the
solvent mixture with varied ratio of ethyl acetate over hexane.

Example 2

Production of EODA from DOD by Heat Treatment

[0039] Conversion of DOD by heat treatment was carried out in 4 ml glass
vial containing 10 mg DOD and 500 μl hexane as solvent. The mixture
was incubated at 90° C. for 24 hours on a heating block
(Barnstead/Thermolyne Type 176000 Dri-Bath). At the end of the treatment,
solvent was evaporated using nitrogen flushing and the reaction product
was dissolved in the mixture of chloroform and methanol (1:1, v/v). For
the study of time-coursed production, vials containing 10 mg of DOD were
heated at 90° C. and withdrawn for analysis after a given time.

Example 3

Analysis of Reaction Products

[0040] Reaction products were analyzed by TLC and quantified by GC
analysis with heptadecanoic acid being an internal standard. The TLC
analysis was developed in a solvent system (toluene:1,4 dioxane:acetic
acid, 79:14:7, v/v/v) and the spots were visualized by spraying the plate
with 50% sulfuric acid followed by heating at 95° C. for 10
minutes. For GC analysis, the sample methylated with diazomethane for 5
min at room temperature was analyzed with ACME 6100 Series Gas
Chromatography System (Younlin Co., Korea) equipped with a
flame-ionization detector and a capillary column (SPB-1®, 15
m×0.32 mm i.d., 0.25 μm thickness, Supelco Inc., Bellefonte,
Pa., USA). GC was run with a temperature gradient of 20° C./min
from 100 to 150° C., 5° C./min from 150 to 200° C.,
and then 0.5° C./min from 200 to 210° C. followed by
holding for 10 min at 300° C. (nitrogen gas flow rate=0.67
ml/min). Injector and detector temperatures were held at 270 and
280° C., respectively.

[0041] Chemical structure of the purified target product was determined by
GC/MS, NMR, FTIR. Electron-impact (EI) mass spectra was obtained with a
Hewlett Packard 5890 GC (Avondale, Pa., USA) coupled to a Hewlett Packard
5972 Series Mass Selective Detector. The column outlet was connected
directly to the ion source. Separation was carried out in a
methylsilicone column (30 m×0.25 mm i.d., 0.25 quadraturem film
thickness) with a temperature gradient of 20° C./min from 70 to
170° C., holding for 1 min at 170° C. and 5° C./min
up to 250° C. followed by holding for 15 minutes (helium flow
rate=0.67 ml/min). 1H-NMR and 13C-NMR spectra were determined
in deuterated chloroform with a Varian-500 spectrometer (Billerica,
Mass., USA), operated at a frequency of 400 and 100 MHz, respectively.
FTIR analysis of the purified compound was run as films on KBr on a
Perkin Elmer Infrared Fourier Transform Model 1750 spectrometer (Perkin
Elmer, Oakbrook, Ill., USA).

[0042] Antioxidant activity was analyzed using
2,2-Diphenyl-1-picryhydrazyl (DPPH) assay according to the reports (22).
Briefly, 50 μl of sample solution in DMSO was added to 200 μl of
200 μM DPPH radical solution in a 96-well plate. L-ascorbic acid and
α-Tocopherol were used as positive controls. After 30 min of
incubation at 37° C., the absorbance at 515 nm was measured. DPPH
free radical scavenging activities was calculated using the equation;
radical scavenging activity
(%)=[1-(A.sub.sample-A.sub.blank)/(A.sub.control-A.sub.blank)]×100.
DMSO was used as a control.

Experimental Result 1

Production and Isolation of Major Product

[0043] Heat treatment of DOD in hexane at 90° C. for 24 hours
yielded a mixture of several products including one major product, which
were analyzed by TLC (Rf=7.2, FIG. 1, B) and GC (peak retention
time=8.2-8.4 min, FIG. 1, A). The major product was purified using a
silica-gel column. The target compound (unknown) was obtained from the
fraction of hexane:ethyl acetate (8:2, v/v). The purified product with
white crystal-like powder was identified as a single major peak with 96%
or higher purity by GC (FIG. 2, A) and as one major spot on TLC analysis
(FIG. 2, B).

Experimental Result 2

Structure determination

[0044] The purified target product was subjected to GC/MS, FTIR and NMR
analysis for structure determination. The electron-impact GC/MS spectrum
and the corresponding proposed structure of the methylated product are
shown in FIG. 3. The mass spectra of the purified product were
characterized by six major peaks in the fragmentogram. Beta-cleavage at
both sides of the furan ring yielded a furan fragment at 95 m/z, and
intense peaks at 193 m/z and 209 m/z represented the ions formed by
beta-cleavage of the furanoid ring toward the methyl and the methylated
carboxyl end, respectively. Evidence of the loss of a methoxy group was
seen at 277 m/z. This GC/MS analysis result was in close agreement with
that of the chemically synthesized 9,12-epoxy-octadeca-9,11-dienoic acid,
except the location of the furan ring being two carbons farther from the
carboxyl group (23).

[0045] Based on the GC-MS analysis result, the structure of the target
product was expected as shown in FIG. 3. As seen in the figure, the
furanoid fatty acid was expected to include an epoxy structure connected
by an oxygen atom between carbon 7 and 10 and have two double bonds
between carbon 7 and 8 and between carbon 9 and 10. The GC-MS spectrum
was in perfect agreement with the structure expected from the fragment
pattern.

[0046] Most of the naturally-occurring furanoid fatty acids are found in
trace amounts in fish. Mostly, the furan ring is substituted by one or
two methyl residues. The length of the side chain is various. It is also
known to be contained in human blood in trace quantity. However, one with
no methyl residue is not known to occur naturally and it is reported that
a small amount can be produced as intermediate when linoleic acid is
treated with lipoxygenase. However, the resulting furanoid fatty acid is
different in the position of the furan ring from that provided by the
present disclosure. That is to say, it has an epoxy structure carbon 10
and 13.

[0047] As described, the novel furanoid fatty acid presented in the
present disclosure is a new substance that has never been reported.
Therefore, the inventors performed further structural analysis by NMR and
FTIR.

[0049] Chemical synthesis of a furan fatty acid without a substituent was
first reported by Lie Ken Jie et al. They reported that some fatty acids
containing isomeric C18 furans were chemically synthesized from
furan through complicated multiple steps using several catalysts (24).
Alaiz et al. also reported that 9,12-epoxy-octadeca-9,11-dienoic acid was
synthesized from ricinoleic acid through several chemical steps using
chemical catalysts (25). However, in the present disclosure, no chemical
catalyst was used. Instead, a single heat-treatment step was enough to
produce the novel furan fatty acid from DOD. This is the first report of
one-step synthesis of a novel furan fatty acid from DOD by heat
treatment. Through database search (NIST MS Search 2.0 and Cambridge Soft
Chem Office ver. 5), the inventors of the present disclosure validated
that EODA was a newly synthesized furan fatty acid. Comparison with other
F-acids revealed no information about the compound of the present
disclosure.

Experimental Result 3

Time-Coursed Production and Antioxidant Activity

[0050] EODA was produced in the same manner as in Example 2. The
production of EODA was monitored while varying the reaction temperature
(see FIG. 6). As seen from FIG. 6, the production of EODA increased as
the reaction temperature was higher and as the reaction time was longer.
The maximum production yield was about 80%. It was confirmed that, to
achieve the maximum production yield, the reaction temperature should be
maintained at 85° C. or higher and the reaction time should be
about 48 hours.

[0051] Thus, the time-coursed production of 7,10-EODA was studied for 96
hours at 90° C. As seen from FIG. 7, the production of 7,10-EODA
increased proportionally with time up to 48 hours and reached plateau
thereafter. The maximum production yield under this condition was 82%.

[0052] Since furan fatty acids have been reported to have antioxidant
activity, the antioxidant activity of 7,10-EODA was determined by DPPH
assay as radical scavenging activity and compared to that of DOD (FIG.
8). The radical scavenging activity of 7,10-EODA increased
dose-dependently, presenting 23% at the highest concentration (100
μg/mL) tested, while DOD did not show any activity. Although the
activity was relatively low when compared to that of α-tocopherol
or ascorbic acid, 7,10-EODA showed a clear radical scavenging activity in
a dose-dependent manner. This finding confirmed the previous assumptions
that F-acids exhibit antioxidant activity. Furan fatty acids are strong
scavengers of hydroxyl radicals, inhibit erythrocyte hemolysis induced by
singlet oxygen, and are found exclusively at the sn1 position of
phosphatidylcholine (26). Diet-derived F-acids are incorporated into the
tissue and blood of mammals, especially into phospholipids where they
partly substitute for polyunsaturated fatty acids (PUFA) (27). Based on
these biochemical and biological studies of F-acids, it is considered
that F-acids are critically important antioxidant materials for mammals
including human. Hence, new finding of a simple way for cost-effective
production of F-acid is meaningful.

[0053] To conclude, the present disclosure provides a simple method for
producing 7,10-EODA from a dihydroxyl fatty acid precursor. Considering
the difficulties in purifying natural furan fatty acids because of easy
attack by peroxyl radicals and small quantity and the complicated
multiple steps for chemical synthesis, the present disclosure provides a
useful way to produce the biologically activity F-acid cost-effectively
in large scale.

[0054] As described, the present disclosure allows production of a new
biologically active furan fatty acid through a one-step heat-treatment
process of 7,10-dihydroxy-8(E)-octadecenoic acid prepared from vegetable
oil by microbial conversion. Considering the difficulties in purifying
natural furan fatty acids because of easy attack by peroxyl radicals and
small quantity and the complicated multiple steps for chemical synthesis,
the present disclosure provides a useful way to produce the biologically
activity F-acid cost-effectively in large scale.

[0055] While the present disclosure has been described with respect to the
specific embodiments, it will be apparent to those skilled in the art
that various changes and modifications may be made without departing from
the spirit and scope of the disclosure as defined in the following
claims.